than compounds with much higher Kms. Compounds primarily cleared by
glucuronidation typically have highKms, and therefore rarely saturate their
clearance mechanism (Williams et al., 2004). In the absence of knowledge ofKm,
a substrate concentration of 1mM can be chosen for the ‘‘substrate depletion’’
approach, as described below. A recent report describes a method for
determination of bothVmaxandKmusing the substrate depletion approach
(Nath and Atkins, 2006). In later development, the substrate concentration can
be optimized to the therapeutic concentration.
Although the aim is to make a semiquantitative assessment that should
approximate thein vivoconcentration, accumulating evidence suggests that
plasma concentrationsin vivodo not necessarily equal liver concentrations. For
example, as mentioned above, pitivastatin is actively taken up into the liver by
the hepatic uptake transporters OATP1B1 and OATP1B3 (Hirano et al., 2004),
which likely results in a high liver:plasma ratio.
Additionally, the role of protein binding to plasma proteins in defining drug
concentrations available to drug metabolizing enzymes in liver microsomes
in vitrois not well understood. Advances in measurement and prediction of
protein bindingin vitrooffer the potential to fill this gap (Obach, 1997; Wring
et al., 2002). As mentioned above, understanding of liver/plasma ratiosin vivo
is still an area for developing further understanding.
Enzyme concentrations in in vitro incubations will determine rate of
turnover. Typical concentrations for human liver microsomes range between
0.5 and 1.0 mg/mL protein. At concentrations above 2 mg/mL the nonspecific
binding of parent drug and metabolites may be too great to yield useful
information. Typical cell concentrations for hepatocytes range from 0.5 to 1.0
million cells/mL (Williams et al., 2003).
15.6.3.2 Substrate Depletion Versus Metabolite Formation In many cases
multiple enzymes will catalyze the formation of multiple metabolites from the
compound of interest, which presents a level of complexity for analysis of
metabolite formation that may not be justifiably resourced in the earlier stages
of development compared to the later stages. The substrate depletion approach
provides a method to determine relative contributions of individual enzymes
using a single analytical assay and appropriate calibration curves for the parent
compound (Williams et al., 2003). This would therefore offer an opportunity to
understand potential intersubject variability in pharmacokinetics of the parent
compound as opposed to variability in the individual clearance pathways. It
also provides a method to determine theKm–Vmax, and therefore the potential
for nonlinear pharmacokinetics (Obach and Reed-Hagen, 2002). A key
assumption of the substrate depletion approach is that the substrate
concentration is well belowKm. Therefore, 1mM is an appropriate default
concentration.
Compounds most suited to the substrate depletion approach are those that
are turned over rapidly. For compounds that are turned over more slowly, for
example those where the depletion of parent is less than 20% over the incubation
492 REACTION PHENOTYPING